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Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp. citri


Xanthomonas citri subsp. citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. The aim of the present study was to describe the recombinant expression, purification and characterization of a cysteine peptidase from Xanthomonas citri subsp. citri (strain 306) that is a candidate potentially involved in the pathogenicity of this bacterium. The gene was cloned and expressed in Pichia pastoris and the cysteine peptidase was successfully expressed, secreted and purified by affinity chromatography with yield of about 10 mg/L. An anti HISCPXAC polyclonal antibody recognized the purified recombinant cysteine peptidase HisCPXAC, confirming the correct production of this protein on Pichia pastoris. The same antibody detected the protein in the culture supernatant of Xanthomonas. citri. subsp. citri grown in pathogenicity-inducer medium. Kinetic analysis revealed that HISCPXAC was able to hydrolyze the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin (Z-Leu-Arg-MCA) substrate, with a catalytic efficiency (kcat/km) of 47 ¼M-1.s-1. The purified HisCPXAC displayed maximal catalytic activity at pH 5.5 and a temperature of 30 °C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64 as well as the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3 and CaneCPI-4, with Ki values of 1.214, 84.64, 0.088, 0.086 and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first five amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies by other authors that identified this sequence in a secreted protein from Xanthomonas spp (AU)

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